RU2698139C2 - Description of microorganisms characteristics using maldi-tof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method of obtaining a region for characterizing an analytic plate for characterizing a population of a microorganism in the presence of an antimicrobial agent by MALDI, a method of characterizing a microorganism population (versions), an analytical plate and a device for characterizing a microorganism population which is said plate. Method of obtaining a characterization zone involves providing an analytical plate with an antimicrobial agent-containing zone, applying a microorganism population thereon, incubating and applying a matrix suitable for the MALDI method to said zone. Method for characterizing a population of a microorganism in one embodiment comprises preparing a characterization zone and verifying the presence of the antimicrobial agent and/or the antimicrobial agent degradation product using MALDI-TOF, and in another version includes a first analysis of the characterization zone using the MALDI method to identify a family, genus or species of the microorganism population and a second analysis of the MALDI characterization zone to determine the presence of a population of the antimicrobial agent-resistant microorganism. First analysis and the second analysis are carried out using separate first and second ionisation stages of the same characterization zone. Analytical plate comprises an antimicrobial agent applied in the form of a solid layer on the analytical zone.

EFFECT: inventions provide an easy-to-implement method of obtaining a region for characterizing a microorganism on an analytical plate for identifying its resistance to the antibiotic and identifying the microorganism itself using the MALDI technique.

25 cl, 6 tbl, 5 ex, 9 dwg

Description

The present invention relates to the field of microbiology. More specifically, the present invention relates to characterizing a population of at least one microorganism using mass spectrometry, and in particular mass spectrometry (MS) with time-of-flight matrix-activated laser desorption / ionization, known as MALDI-TOF.

For several years, the MALDI-TOF method was used to quickly identify the microorganism at the species level. Various types of tools that are suitable for this type of characterization are sold by the applicant, and also, in particular, Bruker Daltonics and Andromas.

The microorganism is identified in MALDI-TOF by the spectrum of the most abundant proteins in the microorganism, compared with the reference data in order to identify the family, genus, and usually the type of microorganism. Typically, the protocol used includes applying at least a portion of the colony of microorganisms to the MALDI plate, adding a matrix suitable for the MALDI method, obtaining a mass spectrum, and identifying the species by comparison with the reference data stored in the database.

Recently, the MALDI method has also been used to detect the phenomenon of antibiotic resistance of a microorganism, and, in particular, to identify the phenotype that is responsible for the hydrolysis of beta-lactam type antibiotics, due to the secretion of beta-lactamase type enzymes, and in particular carbapenemase type . In this regard, may be mentioned applications US 2012/0196309 and WO 2012/023845. Characterization of resistance using MALDI-TOF includes detection of the hydrolyzed form and / or loss of the native form of the beta-lactam antibiotic after incubation with the indicated antibiotic sample, which may contain a microorganism capable of producing beta-lactamases.

Although the preparation of the sample for application is not described in detail in these patent applications, it is generally assumed that the microorganism is mixed with the antibiotic in advance, and then the resulting mixture is applied to the MALDI plate or a microorganism (s) lysate is used (see WO 2012/023845, in particular , paragraph 15).

WO 2012/113699 provides the use of mass spectrometry in order to evaluate the inhibitory effect of a substance on beta-lactamases, with the assessment possibly carried out in the presence of bacteria.

The following publications: (Hrabak, Walkova et al. 2011, Hooff, van Kampen et al. 2012, Hrabak, Studentova et al. 2012, Sparbier, Schubert et al. 2012) provide a more detailed description of the protocols that will be implemented for sample preparation while the protocols include the following stages:

- preparation of an inoculum containing colonies of microorganisms;

- centrifugation of the inoculum;

- resuspension of bacterial granules with an antibiotic solution, with or without reagent for lysis;

- incubation for from 30 minutes to 3 hours;

- centrifugation; and

- transfer 1 μl of the supernatant to the plate for MALDI and add 1 μl of the matrix, analysis using MALDI-TOF mass spectrometry.

Preparing a sample to detect resistance by the MALDI-TOF method is thus long and tedious. In fact, the procedure used to detect resistance by the MALDI-TOF method entails the need to prepare a high concentration inoculum and / or one or more centrifugation steps. These stages require supplies, supplies and special equipment, and thus consume supplies, time and operator experience. In addition, before starting this type of preparation, it is difficult to use the MALDI-TOF method routinely to detect resistance, given that describing the characteristics of a large number of samples per day may not be provided. In fact, often there is a mismatch between the presence of a tool for identifying the phenomenon of stability by the MALDI-TOF method and the presence of samples obtained after the mandatory preparation stage. Another problem is that bacteria are incubated with an antibiotic in volumes of the order of 10 μl - 50 μl, which leads to a potentially high risk of reducing enzyme-antibiotic interactions. As a result, there may be a decrease in the efficiency of enzyme activity, which can affect the sensitivity of detection and, thus, the reliability of the method, in particular with microorganisms that are known to produce or secrete small amounts of enzymes.

The detection of antimicrobial resistance is also described in WO 2011/045544 and US 2012/0196309, as carried out using other mass spectrometry methods such as tandem mass spectrometry (MS-MS) and multiple reaction monitoring (MRM). Under such conditions, analysis by mass spectrometry and, as a result, by ionization is carried out not on a microorganism, but on proteins obtained after various purification operations.

In the context of the present invention, the inventors propose to implement a new method of obtaining a characterization zone for characterizing a microorganism by the MALDI method, which can be used to identify antibiotic resistance and which is easy to implement. In addition, this new method is compatible with obtaining various characteristics of a sample present in the characterization zone of at least one corresponding to the determination of antibiotic resistance, and another, often corresponding identification of the microorganism.

The invention thus relates to a method for characterizing a sample that contains at least one microorganism using the MALDI-TOF method, which can be used to determine whether or not a population of a microorganism is stable, at least , to one antimicrobial agent, and within a relatively short period of time, of the order of several hours to one hour or even less.

The invention also relates to a method for producing a characterization zone of an assay plate in order to perform at least one characterization of a population of at least one microorganism in the presence of at least one antimicrobial agent, wherein said characterization involves mass analysis spectrometry during which a population of at least one microorganism deposited in the analytical zone undergoes at least one stage of ionization by bombarding the laser analytical zone beam according to mass spectrometry with matrix-assisted laser desorption ionization, known as MALDI;

The method for obtaining the analytical zone is characterized in that it includes the following stages in sequence:

- a stage consisting in providing an assay plate for characterization by the MALDI method, where the plate comprises at least one assay zone carrying an antimicrobial agent;

- the stage of applying a population of at least one microorganism in the specified analytical zone in contact with an antimicrobial agent;

- the incubation stage, which consists in maintaining the analytical plate under conditions and for a time sufficient for the interaction of the antimicrobial agent and the microorganism present; and

- the stage of applying the matrix, which is suitable for the MALDI method, on the analytical zone.

Preferably, in the context of the preparation process in accordance with the present invention, a population of one microorganism to be characterized is applied. Thus, the characterization zone can then be used to characterize a single microorganism.

In accordance with the invention, the applied population of the microorganism (s) is obtained without a preliminary step of contact with an antimicrobial agent, i.e. the applied population of the microorganism (s) is not mixed with the antibacterial agent present in the characterization zone prior to application.

The method in accordance with the present invention is mainly carried out on a population of microorganism (s) obtained after the stage of concentration, enrichment and / or purification and / or after growth on a suitable medium, in particular a gel medium corresponding to a colony or fraction of a colony.

As an example, in the context of the present invention, the antimicrobial agent is selected so as to enable the detection of resistance due to the production of beta-lactamase, and in particular due to carbapenemase.

The antimicrobial agent, typically an antibiotic, is preferably selected from among penicillins, cephalosporins, cefamycins, carbapenems and monobactams, and in particular from ampicillin, amoxicillin, ticarcillin, piperacillin, cefalactim, cefoxidim, cefoximetsef cefixit, cefoximit, cefoxitime, cefoxitim, cefoxitim, cefoxitim, cefoxitim, cefoxitim, cefoxitim, cefoximit, cefoximit, cefoximit, cefoximit, cefoximit, cefoximetzite, cefoximetzite, cefoximetzite, cefoximetzite , cefpodoxime, cefepime, aztreonam, ertapenem, imipenem, meropenem and faropenem.

The production method in accordance with the present invention may include a functionalization step to obtain an assay plate for characterization using the MALDI method, containing at least one assay zone carrying an antimicrobial agent, called a functionalized zone, wherein said functionalization step is preferably carried out by applying an aqueous solution antimicrobial agent followed by drying.

The invention also relates to a method for characterizing a population of at least one microorganism, where the characterization includes at least determining the presence or absence of a population of a microorganism that is resistant to at least one antimicrobial agent.

This method is characterized in that it consists of the following stages:

- obtaining at least one characterization zone on the analytical plate in accordance with the production method according to the invention;

- the use of mass spectrometry with matrix-activated laser desorption / ionization, known as MALDI-TOF, to analyze at least once the population of the microorganism deposited on the characterization zone for the purpose of obtaining a conclusion whether the population of the microorganism that is resistant to the antimicrobial agent is present in characterization zone.

In particular, mass spectrometric analysis using the MALDI-TOF method to determine whether a population of a microorganism that is resistant to an antimicrobial agent is present in the characterization zone is to verify the presence of an antimicrobial agent and / or antimicrobial agent degradation products.

Advantageously, the characterization method in accordance with the present invention includes at least two characterizations. In particular, characterization further includes the identification of a family, genus or, preferably, a species of a microorganism population deposited on the characterization zone.

Preferably, the characterization method in accordance with the present invention includes:

- determination of the family, genus, or, preferably, the type of population of the microorganism deposited on the characterization zone by conducting a first analysis by mass spectrometry of the MALDI type corresponding to the first stage of ionization of the characterization zone, and the first calibration for analysis; and

- determination of the possible presence in the characterization zone of a population of a microorganism that is resistant to the antibacterial agent by conducting a second analysis by mass spectrometry using the MALDI-TOF method corresponding to the second stage of ionization of the same characterization zone, and using a second calibration for analysis.

In such circumstances, the identification of a family, genus or, preferably, a species of a population of a microorganism and the determination of the possible presence of an antimicrobial agent preferably refers to the same microorganism.

The invention also relates to a method for characterizing a population of at least one microorganism, where the characterization includes at least:

- identification of the family, genus or, preferably, the type of population of the microorganism, by conducting the first analysis by mass spectrometry using the MALDI method, corresponding to the first stage of ionization of the characterization zone containing the population of the microorganism (s), an antimicrobial agent and a matrix suitable for the MALDI method;

- determining the presence of a population of a microorganism that is resistant to at least one antimicrobial agent, by performing a second analysis by mass spectrometry using the MALDI method, corresponding to the second stage of ionization of the characterization zone, comprising a population of at least one microorganism, an antimicrobial agent and a matrix suitable for the MALDI method;

where the method is characterized in that:

- the first analysis and the second analysis, respectively, are carried out using a separate first stage and a separate second stage of ionization of the same characterization zone, including a population of at least one microorganism, and an antimicrobial agent;

- the first analysis uses the first calibration, and the second analysis uses the second calibration, which is different from the first calibration.

Preferably, the applied population includes one characterized microorganism.

The characterization method of this species preferably uses a method for obtaining a characterization zone of the assay plate according to the present invention.

In the context of this invention, the same characterization zone and the same population of microorganism (s) undergo two ionizations in a row in order to obtain the two desired characterizations: characterization of the phenomenon of resistance of the microorganism to an antimicrobial agent, and identification of the microorganism. The invention also relates to a method for functionalizing the analytical zone of the analytical plate adapted to the MALDI method, where the method for functionalizing the analytical zone is characterized in that it includes:

- the stage of functionalization of the analytical zone, in order to give the analytical zone the ability to take an antimicrobial agent.

The functionalization step can be implemented by applying an aqueous solution of an antimicrobial agent, followed by drying.

The invention also provides an analytical plate for receiving a population of at least one microorganism to be characterized in the analytical zone by mass spectrometry in accordance with the MALDI method, where the plate is characterized in that it includes an antimicrobial agent deposited, in particular, in the form of a solid layer on the specified analytical zone of the analytical plate, thereby forming a functionalized analytical zone. In particular, in this type of assay plate, a functionalized assay zone (or zones) carrying an antimicrobial agent (s) does not include the microorganism in an amount that is detected by the MALDI method and / or a functionalized assay zone (or zones) is dried.

The term “dried”, in particular, means that the antimicrobial agent is not in solution, but in the form of a solid layer that adheres to the analytical zone on which it is applied. In particular, the preservation of the antimicrobial agent in the analytical zone, which undergoes functionalization, can be carried out by electrostatic binding, ionic binding, covalent binding, affinity binding, or in fact using an adhesive agent. In particular, the adhesive agent may be a polymer that is soluble in water. An example of an adhesive agent that can be used to immobilize an antimicrobial agent in an assay zone or zones that can be mentioned is heptakis (2,6-di-O-methyl) -β-cyclodextrin.

In a specific embodiment, on an assay plate intended to produce a population of at least one microorganism to be characterized, the assay zone or each assay zone carries a single antimicrobial agent.

An assay plate for receiving a population of at least one microorganism in accordance with the present invention may be packaged in hermetically sealed packaging. In particular, hermetically sealed packaging may be suitable for protecting the plate from light and moisture.

A functionalized analytical zone (s) can be obtained by applying an aqueous solution of an antimicrobial agent, followed by drying.

Plates of this type may include a plurality of functionalized assay zones, each of which carries an antimicrobial agent, in particular at least a first functionalized assay zone bearing a first antimicrobial agent and a second assay zone containing a second antimicrobial agent that is different from the first antimicrobial agent.

Typically, plates of this kind include at least one reference analytical zone intended for subsequent intake of a population of a reference microorganism, and for this reason, the surface of the reference analytical zone does not contain an antimicrobial agent.

The invention also relates to the use of an assay plate in accordance with the invention in a characterization method in accordance with the present invention.

The following description, with reference to the accompanying drawings, contributes to a better understanding of the invention.

Figure 1 is a schematic top view of a MALDI plate.

Figures 2-4 are mass spectra obtained in the examples using the MALDI-TOF method. In these figures, the intensity scale is a relative scale with reference to the most intense peak of the spectrum. As an example, in the selected mass range (in particular, from 200 m / z to 1200 m / z), if the most intense peak is 100 mV, then it is taken as 100% (as indicated on the left side of the spectra). Less intense peaks are indicated relative to the most intense peak: thus, a peak with an intensity of 75 mV reaches 75% of the scale (on a spectrum that contains a maximum peak intensity of 100 mV). As a result, for the same spectrum, the peak intensity level between the strains cannot be compared. In contrast, these spectra can be used for the same strain to compare the intensities between the native peak of the antimicrobial agent and its degradation product. It is also possible for the same strain to compare the intensity between native or hydrolyzed peaks and a control peak that has not been subjected to changes that induce the biological / enzymatic activity of the test microorganism. The following peaks: the peak of the HCCA matrix, the peak of the peptide or reference molecule added to the matrix, or which has already been dried in the analytical zone, can be considered as control peaks.

The figure 5 shows the appearance of the analytical zones (spots), which was applied antibiotic, Faropenem, in the presence and in the absence of an adhesive agent (heptakis), before and after scraping with an inoculation loop.

Figure 6 shows the change in the ratio of the intensities of the peaks of native faropenem and hydrolyzed faropenem obtained by the MALDI-TOF method compared to the control peak of HCCA depending on the ratios [heptakis] / [faropenem].

The figure 7 presents the mass spectra obtained in the second series of data collection in example 5.

The figure 8 shows the change in the ratio of intensities 304/308 and 304/330 depending on the concentration of the inoculum used for the two strains tested in example 5.

Figure 9 is an embodiment of the case supplemented with a MALDI plate that can be adapted to apply a liquid microorganism population.

MALDI Analytical Plates

The MALDI assay plate has at least one, and generally a plurality of assay areas. Analytical zones are presented in the form of spots, usually round in shape. In order to facilitate subsequent ionization at least at the level of the analytical zone or zones, the surface of the plate is conductive. For example, an analytical plate of this kind is formed by a polymer, such as polypropylene, wherein said polymer is coated with a layer of stainless steel. The polymer may comprise an electrically conductive material such as carbon black. By way of example, such a plate may be a plate marketed by Shimadzu under the name “Fleximass ™ DS disposable maldi targets)).

A wide range of MALDI inserts are commercially available, including bioMerieux Fleximass DS inserts (disposable or reusable) and Bruker Daltonics MALDI Biotarget inserts. Plates I of this kind, as a rule, include from 48 to 96 analytical zones 1 or spots, and at least one, or even two or three reference analytical zones 2, the dimensions of which, as shown in the example in Figure 1, differ from analytical zones.

In the context of the present invention, the term “characterization zone” is used for an analytical zone carrying an antimicrobial agent, a population of microorganism (s), and a matrix suitable for the MALDI method.

Functionalization of analytical zones

The term "antimicrobial agent" means a compound that is able to reduce the viability of the microorganism and / or reduce its growth or reproduction. Antimicrobial agents of this type can be antibiotics when they are directed against bacteria. However, this invention is applicable to any type of microorganism from a number of bacteria, yeasts, molds, or parasites, and thus to the corresponding antimicrobial agents.

Preferably, if the antimicrobial agent is an antibody, such as beta-lactam, in particular selected from penicillins, cephalosporins, cefamycins, carbapenems and monobactams, and in particular from ampicillin, amoxicillin, ticarcillin, piperacillin, cefocalim, cephalotin cefoxitin, cefixime, cefotaxime, ceftazidime, ceftriaxone, cefpodoxime, cefepime, aztreonamoma, ertapenem, imipenem, meropenem and faropenem.

Carbapenems are used, in particular, as a last resort for controlling gram-negative bacteria, such as the Enterobacterium family, Pseudomonas and Acinetobacter. An antibiotic of this type is thus applied to the assay area when it is suspected that the microorganism present is an enterobacterium or other gram-negative species that may be resistant to carbapenems.

Preferably, it is applied from an aqueous solution of an antimicrobial agent.

A buffer suitable for dissolving the antimicrobial agent, as well as for optimized activity of the mechanism in the field of target stability, can also be used to prepare a solution of the antimicrobial agent. You can also include the addition of zinc salts (in particular zinc chloride or sulfate type) to the antimicrobial solution to optimize the activity of metal-beta-lactamases.

A drop, for example, from about 1 to 2 microliters, of the antimicrobial solution can be applied so that the entire analytical area is covered. The water contained in the solution is then evaporated, for example, simply by drying in atmospheric air and at ambient temperature. As an example, the analytical plate can be left at a temperature that is in the range from 17 ° C to 40 ° C, and, in particular, at ambient temperature (22 ° C). In addition, in order to accelerate the drying, you can transfer it to a thermostatic chamber, for example, at 37 ° C.

The antimicrobial agent is applied in an aqueous solution in a very simple way, this application follows the drying operation. Thus, an analytical zone is obtained that carries an antimicrobial agent, which is called functionalized. It is also possible to immobilize the antimicrobial agent in the assay zone using an electrostatic, ionic, covalent or affinity bond, or using an adhesive agent. A simple application of an antimicrobial agent could not ensure satisfactory immobilization. In particular, if the antimicrobial agent does not bind sufficiently to the characterization zone, this can lead to the loss of the antimicrobial agent when applying the population of microorganism (s), which would require some skill on the part of the operator when applying, or, in fact, this can lead to loss of antimicrobial agent due to separation of the layer during storage of the plate for MALDI for subsequent use. In addition, instead of simple application, the antimicrobial agent could be bound to the analysis zone using electrostatic, ionic or covalent bonds with or without the use of an optionally specific linker or “arm” (antibodies, recombinant phage proteins), using biotin / streptavidin, already grafted to the surface of the analytical zone and the antimicrobial agent, or any other type of bond adapted to the nature of the antimicrobial agent and to the surface of the analytical zone, or using adhesive wow agent. Nevertheless, the mode of binding or application should be chosen so that any interaction of the antimicrobial agent with the microorganism is not disturbed, as this can lead to a masking of the resistance phenomenon. In particular, it is preferable to immobilize the antimicrobial agent with an adhesive agent, rather than by covalent binding or binding affinity, in order to prevent changes in the conformation of the antimicrobial agent and to ensure that it can properly access the active site of the enzyme, which can produced by a microorganism.

When an adhesive agent, i.e. an agent that adheres to the plate, and thus improves the immobilization of the antimicrobial agent to it, is used, then a mixture of the adhesive agent and antimicrobial agent in an aqueous solution is applied. In particular, the adhesive agent may be a water soluble polymer. An example that can be mentioned as an adhesive agent that can be used to immobilize an antimicrobial agent in an assay zone or zones is heptakis (2,6-di-O-methyl) -β-cyclodextrin. The adhesive agent should be selected depending on the function of the antimicrobial agent immobilized in the assay area. In particular, it should be selected depending on its mass, so that its presence does not distort the subsequent detection of MALDI in order to determine the presence or absence of the antimicrobial agent present and / or its degradation products. A person skilled in the art should adjust the amount of adhesive agent that should not be too high so that the antimicrobial agent is available to the microorganism population after application of the latter. As an example with heptakis (2,6-di-O-methyl) -β-cyclodextrin, we can choose the mass ratio of heptakis (2,6-di-O-methyl) -β-cyclodextrin to antimicrobial agent from 1/20 to 1/2, preferably from 1/10 to 1/5.

One skilled in the art will adapt the amount of antimicrobial agent deposited on the assay area as a function of the antimicrobial agent in question. In fact, depending on the degree of ionization of the molecule, a sufficient amount must be applied in order to be able to detect peak (s) corresponding to the antimicrobial agent using MALDI-TOF with intensities above background noise. In contrast, too much antimicrobial agent can lead to a risk of masking the detection of a characteristic phenomenon of resistance, so that a decrease in the peak intensity corresponding to the native antimicrobial agent may not be observed. Excess antimicrobial agent may, in particular, compromise the detection of low activity beta-lactamase. For example, 0.04 g / m ² - 4 g / m ² antimicrobial agent should be applied. For this purpose, a solution of the antimicrobial agent in water, in particular in ultrapure water, should be applied in a concentration of from 0.1 mg / ml to 10 mg / ml. For example, in the case of ampicillin, an aqueous solution containing 1.7 mg / ml to 10 mg / ml of ampicillin may be applied, and in the case of pharopenem, an aqueous solution containing 0.1 mg / ml to 1 mg / ml of pharopenem may be applied.

The characterization zone should preferably carry one antimicrobial agent, although the use of multiple antimicrobial agents on the same analytical zone is not excluded. A characterization zone bearing a plurality of antimicrobial agents could be used to check for the presence of multiple enzymes at the same time, and thus for various resistance phenomena. When a characterization zone carrying a plurality of antimicrobial agents is used, the agents must be selected in such a way that the masses of their native form and / or their decomposition products under the action of the target enzyme do not overlap, so that they can be detected separately by MALDI-TOF. For example, you can add another antimicrobial agent, from the same family, or from another family in addition to the first antimicrobial agent. For example, some carbapenems are more capable of identifying a specific carbapenemase. Thus, it is possible to envisage the presence of many types of carbapenems or other beta-lactams in the same characterization zone. In contrast, if two antimicrobial agents are applied to the same area, they should be selected so that their activity spectra do not interfere with each other so that they can be separately detected using MALDI-TOF.

In addition, another compound may be added in addition to antibacterial agents. With beta-lactams, it is also possible to apply a beta-lactamase inhibitor in order to characterize the ESBL phenomenon (extended spectrum of beta-lactamases), as used in particular in WO 2012/023845. In particular, a combination of beta-lactam with a beta-lactamase inhibitor such as clavulanic acid, sulbactam or razobactam may be applied. If the MALDI plate contains many analytical zones, at least two zones, or even more, preferably carry different antimicrobial agents. You can also characterize several populations of the microorganism (s) with a single plate. Each zone can carry a separate antimicrobial agent. Typically, however, characterization is carried out in two repetitions, so that a single antimicrobial agent is present in at least two analytical zones, or even more zones.

Functionalized MALDI plates of this type can be delivered directly to the consumer, who in this case should only apply the studied population of the microorganism (s), and then, after the incubation step, the MALDI matrix. Such plates may be implemented as individual packages or packages containing multiple plates.

Preparation and application of a population of microorganism (s)

In the context of this invention, a population of the microorganism (s) is applied to the assay area of the MALDI Plate functionalized with an antimicrobial agent to characterize it.

A population of microorganism (s) may come from various sources. Examples of sources of microorganism (s) that may be mentioned are samples of biological origin, in particular of animal or human origin. A sample of this type may correspond to a sample of biological fluid, whole blood, serum, plasma, urine, cerebrospinal fluid, or organic secretion, or a sample of tissue or isolated cells. This sample may be applied as is or, preferably, may be subjected to a preparation comprising enrichment or concentration of the culture and / or extraction, or a purification step using methods known to one skilled in the art, before application to the assay area under consideration. However, this type of preparation should not be a lysis, which can cause the breakdown of microorganisms and the loss of their contents before application to the analytical zone. The population of the microorganism (s) may be applied as an inoculum. In the context of the present invention, the population of the microorganism (s) deposited on the assay zone is preferably a population of the living microorganism (s), although the extraction of the population of the microorganism (s) from the biological sample using a detergent that may affect viability is not excluded. However, under such conditions, in order to conduct an immediate test of the population of microorganism (s) using MALDI, the already existing supply of active enzymes can be used to characterize the population of microorganisms (s) by detecting any resistance phenomenon.

The source of the population of the microorganism (s) may also be an agri-food product, such as meat, milk, yogurt, and any other consumed product that may be contaminated, or may actually be a cosmetic or pharmaceutical product. In this case, this type of product may be subjected to enrichment or preparation of the culture, concentration and / or extraction or purification stage to obtain the applied population of the microorganism (s).

As a rule, the source of the microorganism (s) can be preliminarily placed in the culture in broth or on a gel in order to enrich it with microorganisms. Gel or broth carriers of this type are well known to those skilled in the art. Gel enrichment is particularly advantageous since it can be used to obtain colonies of microorganisms that can be applied directly to the assay area.

In the context of the present invention, it is preferable to apply to the assay area a cell medium containing a bacterial population, rather than one or more proteins obtained after the extraction or purification step, as is the case with MS / MS or MRM methods. Preferably, if the applied population of microorganisms contains at least 105 CFU of microorganisms. For example, 10 ⁵ CFU - 10 ⁹ CFU of a microorganism can be applied. For example, you can proceed directly to the application of biomass by dropping a suspension of microorganisms in ultrapure water or buffer. A colony or fraction of a colony of a microorganism may be applied.

The applied population preferably contains one type of microorganism. Nevertheless, application of a population containing various microorganisms to the analytical zone is not excluded. In this case, it is preferable that it is known that the microorganisms will develop various resistance mechanisms so that it is possible to know which microorganism represents the identified resistance.

In the context of this invention, it is not advisable to carry out any preparation of the applied sample. In particular, a population of microorganism (s) is applied without prior contact with an antimicrobial agent. In fact, in the context of the present invention, there is no need to carry out lengthy and tedious preparation of the applied sample; the applied population of microorganism (s) can be obtained without a centrifugation step.

The application is carried out in such a way that the population of the microorganism (s) is applied uniformly to the analytical zone. For this purpose, you can use the procedures used to conduct standard identification of microorganisms described in the manuals for commercial MALDI-TOF tools, such as the VITEK® MS tool manufactured by bioMerieux.

However, in addition to the population of microorganism (s), it is also possible to add a compound that is known to accelerate the enzymatic reaction found in the stability mechanism under consideration. This compound may be zinc, for example, in the form of ZnCl ₂ or zinc sulfate, which, in particular, is an important cofactor of the activity of metal-beta-lactamases. This compound can already be applied to the assay zone in combination with an antibacterial agent or at any other time during the preparation of the characterization zone.

The fact that the applied substance to the assay area does indeed contain a population of at least one microorganism can be determined initially using a suitable test, in particular based on the fact that it is a colony isolated on a gel. Preferably, one population of one microorganism is applied to the assay area.

Incubation

After applying the population of microorganism (s), the analytical zone carrying both the antimicrobial agent and the population of the characterized microorganism is subjected to incubation stages in order to ensure the interaction of the microorganism and the antimicrobial agent, and thus, in the case of the presence of a population of microorganisms, which resistant to antimicrobial agent, manifestations of the reaction / phenomenon of the emergence of resistance. In particular, when the detected phenomenon of resistance is due to the presence of an enzyme produced by the applied microorganisms, incubation can be carried out in such a way as to allow an enzymatic reaction to take place.

In the context of this invention, the phenomenon responsible for antimicrobial resistance thus occurs directly on the MALDI plate, and not before the application of the microorganism (s) already in the presence of the antimicrobial agent on the MALDI plate, as is the case with prior art methods . The phenomenon responsible for antimicrobial resistance occurs in a minimum volume corresponding to the characterization zone. Thus, the dilution problems found in the case of prior art methods are limited.

The conditions and incubation period should be adapted by a person skilled in the art as a function of the characterized phenomenon of resistance.

As an example, the analytical plate can be left at a temperature that is in the range from 17 ° C to 40 ° C, and, in particular, at ambient temperature (22 ° C). In addition, you can transfer it to a thermostatic chamber, for example, at 37 ° C, in order to promote an enzymatic reaction, which may be the cause of this phenomenon of stability.

Humidity should be adapted in such a way as to prevent the drying of the present microorganisms, in particular when the detected resistance phenomenon uses the hydrolysis reaction. The plate can be placed during incubation in a humid atmosphere, at least so that the amount of moisture provided by the directly applied substance containing the population of the microorganism (s) is sufficient.

Under the selected conditions, the incubation time should be sufficient to ensure subsequent detection, using MALDI-TOF MS, of a resistance phenomenon that should be detected, in particular, by the enzymatic reaction of the resistance phenomenon mediated by the enzyme. Incubation is usually carried out for at least 5 minutes, more preferably for at least 20 minutes, and even more preferably for a period of from 45 to 90 minutes.

In the context of the present invention, it has been shown that carrying out an incubation step of this type does not in any way adversely affect the subsequent identification of the microorganism, if such characterization is to be carried out in addition to detecting a resistance phenomenon.

MALDI Matrix Application

In general, the matrices used in the MALDI method are photosensitive and crystallize in the presence of a population of microorganism (s), while maintaining the integrity of the molecules and microorganisms present. Matrices of this type, particularly suitable for the MS MALDI-TOF method, are well known and, for example, consist of a compound selected from: 3,5-dimethoxy-4-hydroxycinnamic acid, a-cyano-4-hydroxycinnamic acid, ferulic acid and 2,5-dioxibenzoic acid. Many other compounds are known to those skilled in the art. There are also liquid matrices that do not crystallize at atmospheric pressure or under pressure. Any other compound that can be used to ionize the molecules present in the characterization zone under the action of a laser beam can be used.

In order to obtain a matrix, a compound of this type is usually dissolved in water, preferably of "ultra-pure" quality, or in a mixture of water and an organic solvent (s). Examples of organic solvents that are commonly used and which may be mentioned are acetone, acetonitrile, methanol, and ethanol. Trifluoroacetic acid (TFA) can sometimes be added. As an example, one example matrix consists of 20 mg / ml synapic acid in a mixture of acetonitrile / water / TFA 50/50 / 0.1 (v / v)). An organic solvent allows the hydrophobic molecules present to dissolve in solution, although water can be used to dissolve hydrophilic molecules. The presence of an acid, such as TFA, promotes the ionization of molecules via proton capture (H +).

The matrix forming solution is applied directly to the assay area and then the population of microorganisms and antimicrobial agent present on it is covered.

Optionally, the method in accordance with the present invention may also include the stage of crystallization of the matrix, which takes place before the stage of ionization of the characterization zone. Typically, the matrix is crystallized, allowing the matrix to dry in ambient air. The solvent in the matrix is thus evaporated, for example, by leaving the analytical plate at a temperature that, for example, is in the range from 17 ° C to 30 ° C, and in particular at ambient temperature (22 ° C) for several minutes, for example, from 5 minutes to 2 hours. This evaporation of the solvent allows the matrix to crystallize, in which the population of the microorganism (s) and the antimicrobial agent are distributed.

Ionization and mass spectrum acquisition

The population of microorganism (s) and antimicrobial agent placed in the MALDI matrix and forming a characterization zone undergo mild ionization.

The laser beam used for ionization can have any wavelength that is favorable for sublimation or evaporation of the matrix. Preferably, an ultraviolet wavelength, or even an infrared wavelength, is used. As an example, this ionization can be carried out using a nitrogen laser emitting ultraviolet (UV) radiation at 337.1 nm.

In the process of ionization, the population of the microorganism (s) and antimicrobial agent undergo laser excitation. The matrix then absorbs light energy, and the restoration of this energy sublimates the matrix, which leads to the fact that the molecules present in the population of the microorganism (s) and in the antimicrobial agent are desorbed, and this brings the material into a state called plasma. In this plasma, an exchange of charges occurs between the molecules of the matrix, microorganisms, and the antimicrobial agent. As an example, protons can be detached from the matrix and transferred to proteins, peptides, and organic compounds present in the characterization zone. This stage can be used to conduct soft ionization of the molecules present without causing their destruction. The population of microorganism (s) and antimicrobial agent then releases ions of various sizes. After that, these ions are accelerated by the electric field and fly freely in the tube under reduced pressure, known as the transit tube. The pressure applied during ionization and during the acceleration of the formed ions, as a rule, is in the range from 10 ^-6 to 10 ^-9 mbar [mbar]. The smallest ions therefore "fly" faster than larger ions, which allows them to separate. The detector is located at the end of the span tube. The flight time (TOF) of ions is used to calculate their mass. Thus, a mass spectrum is obtained, which is the signal intensity corresponding to the number of ionized molecules of the same mass per charge (m / z), depending on the ratio m / z of the molecules entering the detector. The mass to charge ratio (m / z) is expressed in Thomsons [Th]. After introduction into the mass spectrometer, the spectrum of the characterization zone is obtained very quickly, usually in less than a minute.

The MALDI-TOF mass spectrometry method suitable for use in accordance with the present invention, in particular, may include the following successive steps to obtain a mass spectrum:

- providing a characterization zone containing a population of microorganism (s) for study and at least one antimicrobial agent in a matrix adapted for MALDI spectrometry;

- optionally, crystallizing the matrix into which the population of the microorganism (s) and the antimicrobial agent are placed;

- ionization of a mixture of a population of microorganism (s) and antimicrobial agent and matrix using a laser beam;

- acceleration of ionized molecules obtained due to potential difference;

- the implementation of the free movement of ionized and accelerated molecules in the tube under reduced pressure;

- detecting at least a portion of the ionized molecules at the exit of the tube so as to measure the time it took them to move through the tube under reduced pressure and to obtain a signal corresponding to the number of ionized molecules reaching the detector at a particular point in time; and

- calculating the ratio of mass to charge (m / z) of the detected molecules so as to obtain a signal corresponding to the number of ionized molecules of the same mass to charge (m / z) as a function of the ratio of m / z of the detected molecules.

In general, the m / z ratio is calculated taking into account the initial calibration of the mass spectrometer, used as an equation relating the mass to charge ratio (m / z) and the transit time of ionized molecules in the tube under reduced pressure.

Calibration involves the use of a molecule or microorganism (depending on characterization) that produce ionized molecules that span the mass range corresponding to the intended characterization. The m / z ratios of these ionized molecules serve as standards, allowing the instrument to properly measure the masses.

To identify the microorganism, calibration can be carried out using a strain of bacteria with ionized molecules that have m / z ratios that span the mass range used for identification (typically in the range of 2,000 daltons (Yes) to 20,000 daltons for yeast, mold, bacteria , or parasites). To detect signals corresponding to an antibacterial agent, calibration can be performed using a mixture of small mass peptides. In the context of the present invention, for example, a pepMIX 6 calibrator (LaserBio Labs) covering a mass range of 350 Da to 1000 Da can be used.

To obtain a mass spectrum, any type of MALDI-TOF mass spectrometer can be used. Spectrometers of this type include:

i) an ionization source (generally a UV laser) for ionizing a mixture of a population of microorganisms (s) and an antimicrobial agent and matrix;

ii) an accelerator of ionized molecules using a potential difference;

iii) a reduced pressure tube in which ionized and accelerated molecules move;

iv) a mass analyzer designed to separate the molecular ions formed depending on their mass to charge ratio (m / z); and

v) a detector designed to measure the level of a signal emitted directly by molecular ions.

In the context of the present invention, analysis using MALDI-TOF is preferably a simple MALDI-TOF analysis, although analysis using MALDI-TOF TOF is not excluded. MALDI-TOF-TOF analysis, although it is more complex, can be provided, in particular, with the aim of increasing the detection sensitivity, under certain circumstances, and this requires a tool that is suitable for this type of analysis.

Antimicrobial Resistance Detection

The term "resistance" means a phenomenon in which a microorganism does not show a decrease in its viability or a decrease in its growth or reproduction when it is exposed to a concentration of an antimicrobial agent that is recognized as effective against said microorganism in the absence of resistance.

The stability mechanism can be identified from the mass spectrum obtained for the characterization zone under consideration by detecting a peak with a given mass on the resulting mass spectrum or a change in the mass peak compared to the reference mass spectrum, in particular, compared to the mass spectrum of the antimicrobial agent present in the specified characterization zone.

In the context of the present invention, it has been shown that mass spectrometry using MALDI-TOF directly on the microorganism in the presence of an antimicrobial agent should allow the detection of molecules of interest that are relevant to determining resistance to said antimicrobial agent. Determining any resistance of a microorganism population in this way may include the following steps:

a1) obtaining a reference mass spectrum, for example, for an antimicrobial agent, and / or for its degradation products; degradation products of this type are the result of the phenomenon of stability and are caused, in particular, by the presence of a degrading enzyme;

b1) the application of ionization to a population of microorganism (s) and antimicrobial agent, which are applied to the analytical zone and applied in the presence of a matrix (corresponding characterization zone in accordance with the invention);

c1) obtaining a mass spectrum after this ionization, in the range of masses of interest, for determining stability; and

d1) comparing the mass spectrum obtained in step c1) with a reference spectrum and deriving from it the presence or absence of stability.

In stage d1), for example, if, on the mass spectrum obtained in stage c1), the peak or peaks with the mass characteristic of the antimicrobial agent have disappeared, and / or if one or more of the peaks with the mass characteristic of one or more decomposition products of an antimicrobial agent are present, it can be concluded that there is a microorganism that is resistant to an antibacterial agent. For example, an interpretation can be made from the ratio of intensities between a peak with a characteristic mass for an antimicrobial agent or one of its degradation products and a peak with a characteristic mass for an external calibrant, or from the ratio of intensities between a peak with a characteristic mass for an antimicrobial agent and a peak with a characteristic mass for the degradation product of an antimicrobial agent.

In addition, it is possible to compare the intensity level between the peak or peaks with the characteristic mass for the antimicrobial agent or the peak or peaks with the mass characteristic of one or more decomposition products of the antimicrobial agent and one or more peaks of the reference compound that is present and which has not been subjected to variations induced by test

biological / enzymatic activity. Examples of reference peaks that may be considered are one or more peaks of the MALDI matrix, one or more peaks of a peptide or reference molecule added to the matrix or that are already dried in the assay zone, or one or more peaks corresponding to a molecule of the microorganism present (e.g. , metabolite), which is always present and unchanged in several forms.

When determining stability, calibration is performed in the mass range corresponding to low masses, typically in the range of 200 daltons to 1200 daltons, and preferably in the range of 200 Da to 600 Da. The mass spectrum obtained in step c1) is also included in this mass range. In order to perform this calibration, two μl of a calibration solution consisting of a mixture of peptides (reMHXB, LaserBio Labs) and an HCCA matrix, α-cyano-4-hydroxycinnamic acid, can be applied, for example, to the reference analysis zone. Before the characterization zones are ionized, a calibrant is ionized in this reference analytical zone. The m / z ratios of the ionized calibrant molecules then serve as standards in order to allow the instrument used to measure the masses appropriately.

The method in accordance with the invention, in particular, can be used to detect resistance due to the ability of the microorganism to secrete an enzyme that is known to degrade beta-lactam-type antibiotics, and in particular selected from penicillinases, cephalosporinases, cefamycinases, and carbapenemase The invention can also be used to detect other stability phenomena based on degradation or enzymatic modification, causing a change in the mass of the antimicrobial agent. As an example, stability mechanisms can be mentioned, such as degradation of macrolides by esterases or degradation of phosphomycin by epoxidases, acetylation of aminosides, chloramphenicol or actually streptogramines, phosphorylation of aminosides, macrolides, rifampicin, and peptide antibiotics, tetracycline hydroxylation, adenylation of rhinamines, and aminylation of rhymes and aminos and glycosylation of macrolides and rifampicin.

The term "degradation product" includes any product corresponding to a change in the chemical structure of the antimicrobial agent due to the action of the microorganism present. In addition to the degradation and enzymatic modification mechanisms mentioned above, the term may also include the addition of a group that is found in MALDI-TOF, which inactivates the antimicrobial agent, or which prevents its binding to its target.

A method of this type for detecting stability can be performed with pre-functionalized MALDI plates using commercially available MALDI-TOF tools. Only the calibration and interpretation steps need to be adapted in order to enable stability detection. In the context of the present invention, it is now possible to detect resistance to antimicrobial agents, and, in particular, a quick determination of resistance to this antibiotic in the usual manner, which can be vital in many clinical cases.

The characterization method in accordance with the present invention, which can be used to identify the presence of a microorganism that is resistant to antibiotics, in a very short period of time is of particular interest for rapid diagnosis. This is especially true for the detection of carbapenemase-producing enterobacteria (CPE). The method in accordance with the invention, can be used for rapid diagnostics in a hospital, in order to adapt the treatment with antibiotics, which are administered in a quick manner.

Microorganism identification

Microorganisms that can be identified using the method according to the invention are all types of microorganisms, pathogenic or otherwise, which are found both in industry and in clinical cases, and which may exhibit the phenomenon of resistance to antimicrobial agents. They can be preferably bacteria, molds, yeast, or parasites. The invention has particular application to the study of bacteria. Examples of microorganisms of this type that may be mentioned are gram-positive, gram-negative, and mycobacteria. Examples of genera of gram-negative bacteria that may be mentioned are: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Acinetobacter, Citrobacter, Aeromonas, Stenotrophomonas, Morganella, Enterococcus and Providencia, and Colliena, and Enterobacter cloacae, Enterobacter aerogenes, Citrobacter sp., Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Providencia rettgeri, Pseudomonas putida, Stenotrophomonas maltophilia, Acinetobacter baumanii, Morumanii, Comumanii, Comomanii, Moromii, Comomilla, Moromanii, Comomilla, Moromanii, Comomilla, Morom senftenberg, Serratia marcescens, Salmonella typhimurium, etc. Examples of genera of gram-positive bacteria that may be mentioned are: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listeria and Clostridium.

Reference spectra obtained using MALDI-TOF for microorganisms of the type that correspond to their basic proteins are available and stored in databases attached to commercial MALDI-TOF instruments, which allows the identification of the presence of such microorganisms, which will be determined by comparison.

Identification of the presence of a microorganism population, thus, may include the following stages:

A2) obtaining a reference mass spectrum of at least one microorganism, and, as a rule, for a number of microorganisms;

b2) applying ionization to a population of microorganism (s) and an antimicrobial agent deposited on the assay zone and in the presence of a matrix (corresponding to the characterization zone in accordance with the invention);

c2) obtaining a mass spectrum obtained after this ionization in the range of masses of interest to identify the microorganism; and

d2) comparing the mass spectrum obtained in step c2) with a reference spectrum and establishing on its basis a family, genus or, preferably, a species of at least one microorganism.

When identifying microorganisms, calibration is carried out in the mass range corresponding to large masses, usually in the range from 2000 Da to 20,000 Da, and preferably in the range from 3000 Da to 17000 Da. The mass spectrum obtained in step c2) is also included in this mass range.

.Calibration can be performed by placing the population of the reference microorganism in the analytical zone present on the plate and its analysis using MALDI-TOF. For example, a reference microorganism of this type may be an E. coli bacterium. For this calibration, you can use the procedures described for standard identification of microorganisms in the operating instructions for commercial MALDI-TOF instruments, such as VITEK® MS manufactured by

.

Two reference analytical zones can be used for calibration: one to identify microorganisms and the other to characterize resistance. In addition, you can use the same reference area to perform both calibrations. In such circumstances, the reference zone must be functionalized with an antibacterial agent, the resistance to which must be studied.

In the context of this invention, it is preferable, but not essential, when both of the following characterizations should be carried out: determination of resistance and identification of the microorganism, then resistance is detected later, that is, the stages of ionization and analysis necessary to detect resistance are carried out in the characterization zone after the stage necessary to identify the microorganism.

When two characterizations are performed on the same zone, this can be done without removing the assay plate from the mass spectrometer used for the MALDI-TOF analysis. This dual characterization, therefore, may include the following stages:

A3) obtaining a reference mass spectrum 1 of at least one microorganism and, as a rule, for a number of microorganisms, and mass spectrum 2 for an antimicrobial agent, and / or for its degradation products;

b3) calibrating the mass spectrometer used with a calibrator used for identification that has already been applied to the reference assay area;

c3) applying ionization to a population of microorganism (s) and antimicrobial agent deposited on the analytical zone in the presence of a matrix (corresponding to the characterization zone in accordance with the invention);

d3) obtaining the mass spectrum obtained after this ionization in the range of masses of interest for the identification of microorganisms;

e3) calibrating the mass spectrometer used a second time with a calibrator used to detect stability that has already been applied to the reference analytical area;

f3) applying ionization again to the population of microorganism (s) and antimicrobial agent deposited on the assay zone and brought into interaction with the matrix (corresponding to the characterization zone in accordance with the invention);

g3) obtaining a mass spectrum obtained after this ionization in the range of masses of interest to determine stability;

h3) comparing the mass spectrum obtained in step d3) with a reference spectrum or spectrum 1 and establishing from it a family, genus or, preferably, a species of at least one microorganism present; and

i3) comparing the mass spectrum obtained in step g3) with reference spectrum 2 and establishing from it the presence or absence of stability.

Steps c3) and f3) are thus carried out in the same characterization zone and thus in the same population of microorganism (s), which thus means that the preparation and manipulation stages can be shortened.

In the context of this invention, the same population of microorganism (s) and its single layer on one analytical zone are used to obtain a characterization zone to which two ionizations are applied successively to conduct two characterizations (characterization of the phenomenon of microorganism resistance to an antimicrobial agent and identification of a microorganism )

In fact, it was unexpectedly shown that the presence of an antimicrobial agent and the preliminary incubation step necessary to detect the resistance phenomenon in no way impede the possibility of identifying the microorganism.

The comparison steps h3) and i3) can be carried out at the end of the method or after each case of the data acquisition under consideration.

It is preferable that the applied population correspond to the population of one microorganism, and two characteristics: detection of resistance and identification of the microorganism, refer to the same microorganism.

In conclusion, the invention can be used to:

- quickly receive information from the same characterization zone and, in particular, characterize the phenomenon of resistance to an antimicrobial agent on the one hand, and identify the microorganism on the other hand;

- simplification of the methods of analysis of MALDI-TOF without lengthy and tedious preparation of the sample before applying it to the MALDI plate to characterize the phenomenon of resistance to antimicrobial agents;

- obtaining optimal contact directly on the MALDI plate of the microorganism and antimicrobial agent sometimes has a positive effect on the sensitivity of the detection of the resistance phenomenon;

- achieving a reduction in time and money as regards consumables and manual work when characterizing the phenomenon of resistance to an antimicrobial agent using MALDI-TOF; and

- the achievement of improved tracking and better control of the analyzed samples, given that there is no need for preliminary incubation in the presence of an antimicrobial agent before applying it to the MALDI plate.

. Анализы проводились в каждом из приведенных ниже примеров в конце сбора данных. Получали спектры для всех зон характеризации, а затем анализировали:The following examples are intended to illustrate the invention, but they are not limited in any way. Analyzes were performed using the VITEK® MS instrument manufactured by the company

. Analyzes were performed in each of the examples below at the end of the data collection. Spectra were obtained for all characterization zones, and then analyzed:

for identification, the spectrum was analyzed using the VITEK MS processor and the V2.0.0 database contained in the Spectra Identifier software version 2.1.0;

for hydrolysis, the peaks of the spectra were visualized using Launchpad V2.8 data acquisition software. Example 1

Preliminary experiments were performed to show that using MALDI-TOF it is possible to detect peaks of beta-lactam type antibiotics after applying a drop of a solution containing an antibiotic mixed with the corresponding matrix for the MALDI method to the analytical zone of the MALDI plate. In the present experiment, the analytical area of the MALDI plate (VITEK® MS disposable TO-430 plates) was treated with a beta-lactam antibiotic and tested to evaluate the ability of antibiotics to be cleaved with beta-lactamase. Ampicillin was used as a model of beta-lactam antibiotic. The experiment was carried out by carrying out the following steps:

- 2 μl of a solution of ampicillin at a concentration of 10 mg / ml in water was applied as a drop on the analytical area of the MALDI plate. The plate was incubated at 37 ° C until the droplet completely dried.

- 2 μl of recombinant beta-lactamase (beta-lactamase Pseudomonas aeruginosa, Sigma-Aldrich lot L6170-550UN CAS: 9073-60-3.).

- 2 μl of recombinant beta-lactamase at a concentration of 1 mg / ml in water, which was used with the addition of zinc in the form of zinc sulfate (0.76 mmol (mm)) to stimulate the enzymatic activity, was applied to a dry assay zone carrying ampicillin. At the same time, a negative control was carried out by applying to another analytical zone treated in the same way with amipicillin, 2 μl of a drop of water with 0.76 mm zinc, but without enzyme.

- The plate was incubated at ambient temperature for one hour to conduct an enzymatic reaction.

- 1 μl of a matrix of HCCA, alpha-cyano-4 hydroxycinnamic acid (matrix VITEK MS CHCA, Biomerieux reference: 411071), was applied to an assay zone already containing ampicillin with or without recombinant beta-lactamase.

- MALDI-TOF mass spectrometric analysis was performed using a VITEK® MS instrument using 2 μl of a mixture of HCCA and reMGX 6 (purchased from LaserBio Labs) used as a calibrant that were applied to a reference assay area in order to calibrate the instrument for low masses. The indicated mixture was obtained by adding one volume of pepMix6 (10X) to 9 volumes of HCCA. A solution of pepMix6 (10X) was obtained in a diluent (trifluoroacetic acid, 0.01% in ultrapure water), in accordance with the manufacturer's recommendations. Peaks were obtained for a low mass range from 200 Da to 1200 Da and studied the presence of molecules corresponding to the native ampicillin molecule or its degradation products after hydrolysis.

All samples were analyzed on a plate in duplicate.

Figure 2 shows the dependence of the obtained mass spectra and shows that ampicillin was not stable on the plate, and that beta-lactamase degrades it effectively after incubation. In fact, in Figure 2, the main peak was observed at 368.09 m / z, which corresponds to the hydrolyzed form of ampicillin (calculated mass of the monosodium form of ampicillin, corresponding to 368.4 m / z) and a complete loss of the native form at 372.09 m / z (mass, calculated for the monosodium form of ampicillin, corresponding to 372.4 m / z) in the upper spectrum, corresponding to the analytical zone with beta-lactamase. In contrast, for the negative control (lower spectrum), the main peak was at 372.09 m / z, which corresponds to the monosodium native form of ampicillin. Slight spontaneous hydrolysis of ampicillin was also observed in the negative control. This indicates that the enzymatic hydrolysis of beta-lactams can be detected using the MALDI-TOF method, using the method of obtaining characterization zones in accordance with the present invention.

Example 2

This experiment was conducted to study the degradation of ampicillin by various strains of bacteria after applying the colonies directly to the analytical zone with ampicillin, which was applied in the form of a solution and dried, as described in example 1.

Bacterial strains and their characteristics are presented in table 1 below.

* MIC: minimum inhibitory concentration of strain

** Virulence factor: corresponds to a known secreted enzyme

The tests were implemented by performing the steps below:

- 2 μl of an aqueous solution of ampicillin at a concentration of 10 mg / ml, with the addition of zinc (0.76 mm), was applied to the analytical zones of the MALDI plate (same as in example 1).

- The plate was incubated at 37 ° C until then, until the drops of the precipitated solution did not completely dry.

- For each bacterial strain, part of the colony obtained by culturing for 24 hours on a gel medium was applied in duplicate to analytical zones containing ampicillin. Each spot was obtained as described in the VITEK® MS procedure for identifying microorganisms.

- The plate was incubated at room temperature (20-25 ° C) for one hour in a humid atmosphere. For this purpose, a closed vessel containing water was used as an incubation chamber for the plate.

- 1 μl of the HCCA matrix was added to the analysis zones bearing both antibiotic and bacteria.

- MALDI-TOF mass spectrometric analysis was carried out using a VITEK® MS instrument, and the plate was installed in vacuum after being placed in the instrument chamber, by performing the following two steps:

- the first acquisition of spectrum data of characterization zones after calibrating the instrument against a reference zone that carries the applied E. coli strain (E-ca1);

- second data acquisition from the same characterization zones after calibrating the instrument for low masses using pepMIX 6 as a calibrant; this second preparation was carried out immediately after the first, without violating the vacuum of the chamber, and without removing the plate from the device.

- Data was collected and compared with the data contained in the database in order to identify the species.

- Peaks were scanned in the low-mass range to detect a native or hydrolyzed form of the antibiotic.

All samples were analyzed on a plate in duplicate.

Figure 3 shows the mass spectra obtained in the second series of data, and it was shown that ampicillin-resistant strains were able to hydrolyze ampicillin under the experimental conditions used, while no degradation of ampicillin was observed in the case of E. coli strain, ATCC 25922, which is known to be sensitive to ampicillin. In fact, after the second acquisition of data at low masses, the main peak at 372.15 m / z, corresponding to the native form of ampicillin, was observed for the sensitive strain and negative control, while for all ampicillin-resistant strains, the main peak was located at 368 , 15 m / z, which corresponds to the hydrolyzed form of ampicillin and the disappearance of the native form.

After the first data acquisition and obtaining the corresponding spectrum, all bacterial strains can be identified with a high degree of confidence.

As can be seen in table 2 below, bacteria can be identified correctly using MALDI-TOF from the first series of data for all characterization zones with a confidence level that lies between 99.9% -100%, showing that the experimental conditions allow to distinguish different kinds.

Example 3

Other tests were performed in order to demonstrate that the determination of the species and the detection of carbapenemase production was possible using the same characterization zone. For this purpose, we used Faropenem as a model of carbapen antibiotic and used a protocol identical to the protocol in Example 2. A solution of Faropenem was prepared at a concentration of 0.5 mg / ml.

Used bacterial strains are described in detail below in table 3.

Figure 4 shows the mass spectra obtained during the second series of data acquisition, and it is shown that the activity of carbapenemase can be detected using MALDI-TOF under these conditions using the second series of data acquisition. Although hydrolyzed forms of faropenem were not detected in this experiment, it was possible to demonstrate the loss of two native forms of faropenem due to the production of carbapenemase by bacteria.

After obtaining data in the range of small masses, for the sensitive strain, the main peak was observed at 308.13 m / z with a small peak at 330.13 m / z, which corresponds to two native forms of faropenem (corresponds to the calculated masses of 308.3 and 330.3 m / z). These two peaks were lost when Faropenem was incubated with the S. marcescens strain producing IMP-1 carbapenemase.

After the first acquisition of data and spectra, both strains can be correctly identified with a high degree of confidence. Similarly, as in Example 2, the presence of faropenem in the characterization zones does not affect the ability of MALDI-TOF to identify species and the ability to distinguish between the type of E.coli bacteria and the type of S. marcescens bacteria, as shown in the results obtained using the first series of data presented in table 4.

Example 4

This test was performed to increase the adhesion of antibiotics to the analytical area of the MALDI plate. In fact, drying the antibiotic solution deposited on the plate led to the formation of a film that is weakly adhered to the surface. Heptakis (2,6-di-O-methyl) -β-cyclodextrin (heptakis, Sigma-Aldrich Ref. H0513) was used to attach faropenem to the surface of the MALDI plate.

In addition to its adhesive properties, the choice of using heptakis was due to its molecular weight of 1331.36 g per mole (g mole ^-1 ), which does not interfere with the mass peaks for faropenem or for its hydrolyzed products. During this experiment, the ratios of the two masses [heptakis] / [faropenem] were tested in order to evaluate the adhesion of dry faropenem and the effect of heptakis on the detection of native peaks and hydrolyzed peaks of faropenem.

Used bacterial strains are described in detail below in table 5.

The tests were implemented by performing the steps below:

- 2 solutions containing a mixture of heptakis and Faropenem and 1 solution of Faropenem without heptakis were prepared in NaCl buffer (0.45%) supplemented with zinc (zinc sulfate, 0.76 mmol). The final concentrations of heptakis and faropenem in these solutions were as follows:

- 0 mg / ml heptakis and 1 mg / ml faropenem;

- 0.1 mg / ml heptakis and 1 mg / ml faropenem (mass ratio 1:10);

- 0.2 mg / ml heptakis and 1 mg / ml faropenem (mass ratio 1: 5);

- 2 μl of each solution was applied to the analytical zones of the MALDI plate (analogously to example 1);

- the plates were incubated at room temperature until the droplets of the precipitated solution completely dried out;

- to assess adhesion, each analytical zone, functionalized using a faropenem or heptakis / faropenem, was scraped off using an inoculation loop in order to simulate the application of a colony, and this slide was photographed;

- in order to evaluate the effect of heptakis concentration on the detection of peaks of faropenem, a part of the colony was obtained after 24 hours cultivation on a gel medium, applied in four replicates to analytical zones bearing separately the pharopenem or mixtures of pharopenem and heptakis;

- the plate was incubated at 37 ° C for 2 hours in a humid atmosphere;

- 1 μl of the HCCA matrix was added to the assay zones, which also carry an antibiotic or a mixture of heptakis / antibiotic and bacteria;

- analysis using MALDI-TOF mass spectrometry was performed using the VITEK® MS instrument, in order to obtain low mass spectra;

- peaks of the native form were observed at 308.3 m / z (Faropenem + Na) and the hydrolyzed form of Faropenem at 304.3 m / z (hydrolyzed Faropenem + H), and the peak for HCCA at 212.03 m / z;

- for all test conditions, the intensities of the three peaks were recorded and the ratios 308/212 and 304/212 were calculated. The peak for HCCA at 212 m / z was used in this example as a control peak of constant intensity.

All samples were analyzed on a plate in four repetitions.

The figure 5 shows the appearance of the analytical zones (spots) before and after scraping using an inoculation loop. The picture shows good dispersion after scraping the heptakis / faropenem mixture over the entire spot surface for [heptakis] / [faropenem] ratios 1/10 and 1/5, with good stability after scraping. The antibiotic, which was dried without heptakis, was not very stable on the surface of the slide and the film completely or partially detached during scraping with an inoculation loop.

Figure 6 shows the change in the ratio of the intensities of the peaks of native faropenem and hydrolyzed faropenem compared with the control peak of HCCA depending on the ratios [heptakis] / [faropenem] used in drying the antibiotic on the slide. Values are the average of four spots. For ratios 1/10 and 1/5, detection was comparable to a state without heptakis, with the added benefit of reproducibility in the presence of heptakis. In fact, significant variability observed in the absence of heptakis (a large margin of error) was associated with complete or partial loss of antibiotic on some spots during colony application. As for the appearance of the hydrolyzed peak after incubation with cattle carbapenemase producing strain, we observed the same phenomenon, i.e. good detection at ratios [heptakis] / [faropenem] 1/10 and 1/5.

The results of this experiment mean that using heptakis in suitable concentrations (0.1 mg / ml or 0.2 mg / ml for 1 mg / ml of pharopenem), the antibiotic can be stabilized on a storage slide, while providing optimized mixing with bacteria in colony application progress. At the same concentrations, heptakis can also be used to improve the reproducibility of results between spots and does not interfere with the detection of peaks or, in fact, the hydrolysis of faropenem.

Example 5

This test was carried out in order to evaluate the possibility of carrying out a hydrolysis reaction in a liquid coating on a functionalized analytical zone of a MALDI plate, i.e. in the bacterial inoculum. In fact, the problem that occurs during colony application is the lack of standardization regarding the number of applied microorganisms, and the variability of the coating depending on the operator. Thus, applying a colony to a slide for normal use in identification using MALDI-TOF requires technical training, and even this does not completely eliminate the risk of variability in results due to heterogeneous coatings. In addition, a bacterial inoculum with a known concentration was applied to the functionalized analytical zones of the MALDI plate.

Used bacterial strains are presented in table 5 above.

The tests were implemented by performing the steps below:

- 2 μl of a solution of pharopenem at a concentration of 1 mg / ml was prepared in NaCl buffer (0.45%) with the addition of zinc (zinc sulfate, 0.76 mmol) was applied to the analytical zones of the MALDI plate and dried at ambient temperature;

- 2 μl of bacterial inoculum at a concentration of 3 units of Mac Farland or 6 units of Mac Farland was applied to the functionalized analytical zones in four repetitions;

- the plate was incubated at 37 ° C for 2 hours in a humid atmosphere;

- to the analytical zones carrying both antibiotic and bacteria, 1 μl of the HCCA matrix was added;

- using the VITEK® MS instrument, two MALDI-TOF mass spectrometric analysis data were collected: one for small mass spectra to observe peaks of pharopenem, and the other for large mass spectra for identification (in accordance with Example 2); and

- peaks were observed for native forms at 308.3 m / z (Faropenem + Na) and at 330.3 m / z (Faropenem + 2Na), and a peak for the hydrolyzed form at 304.3 t / g (hydrolyzed Faropenem + H) ;

- for all test conditions, the intensities of the three peaks were recorded and the ratios 304/308 and 304/330 were calculated in order to quantify the hydrolysis of faropenem.

All samples were analyzed on a plate in four repetitions.

The figure 7 shows the dependence of the mass spectra obtained during the second series of data, and it is shown that the activity of carbapenemase can be detected using MALDI-TOF after applying the bacterial inoculum with a force of 6 units of Mac Farland. In fact, it was possible to demonstrate the loss of two native forms and the appearance of a hydrolyzed form of faropenem due to the production of carbapenemase by bacteria.

In the obtained data, for small masses of the sensitive strain, the main peak was observed at 308.03 m / z and a slight peak was observed at 330.02 m / z, which corresponds to two native forms of faropenem (according to the calculated masses of 308.3 and 330.3 m / z). These two peaks were lost during incubation of Faropenem with a strain of K. pneumoniae producing cattle carbapenemase, and a peak was observed at 304.06 m / z, which corresponds to the hydrolyzed form of Faropenem (estimated weight 304.03).

After the first acquisition of data and spectra, two strains can be correctly identified with a high degree of confidence. The presence of pharopenem in the characterization zones and the inoculum application does not affect the ability to identify species using MALDI-TOF, and does not affect the ability to distinguish between E. coli bacteria and K. pneumoniae bacteria, as can be seen from the results obtained using the first production series data presented in table 6.

The figure 8 shows the change in the ratio of intensities 304/308 and 304/330 depending on the concentration of inoculum used for 2 test strains. For the wild-type strain that does not hydrolyze the antibiotic, no significant differences in the 2 ratios were observed regardless of the concentration of the bacterial inoculum. For a strain producing cattle carbapenemase, a significant increase was observed in both ratios, the value of which is proportional to the inoculum concentration. This increase in ratios led to a decrease in the intensity of the peaks of the native peaks and to an increase in the intensity of the hydrolyzed peak, as shown in FIG. 7.

The results of this experiment show that deposition of the inoculum is also adapted to the hydrolysis reaction on the MALDI plate and to identification using MALDI-TOF mass spectrometry. In addition, the small error bars observed above the average for four applications suggest very good reproducibility of the results.

Figure 9 shows an embodiment comprising a MALDI plate that can be adapted to apply a population of microorganisms in liquid form. A slide of a MALDI plate with assay areas that were already covered with a dried antibiotic was placed in a gallery, inside which many holes were formed (12 in the model shown). Six holes aligned on one axis contained dehydrated opacity controls, and the remaining six holes were empty. The gallery containing the slide was placed in a cassette with a lid. For storage, this cassette can, in particular, be packaged in a hermetically sealed form and protected from light and moisture.

The protocol for using this product can be described in the following steps:

- remove the cover from the cartridge;

- fill 12 wells with a volume of 50 μl - 100 μl of water or physiological buffer. Filling six wells containing opacity control caused the formation of cloudy solutions;

- prepare the inoculum in six other wells by adding a colony or part of the colony in order to obtain a turbidity comparable to the control of the opacity of the anterior well;

- apply 2 μl of each inoculum to the analytical zones bearing the antibiotic;

- add water to the cassette using a pipette to obtain a moist atmosphere;

- replace the lid and incubate at 37 ° C for 1 hour to 2 hours (or less);

- add 1 μl of the HCCA matrix to the analytical zones carrying both antibiotic and bacteria; and

- Replace the slide cover for analysis with MALDI-TOF mass spectrometry.

Adding opacity control means that you can avoid preparing the inoculum using a device for measuring density or turbidity, and it is thus possible to save time. In addition, a measuring device for routine use, such as a densitometer, is adapted for large volumes (1-3 ml), which requires the use of a large number of bacteria.

The inoculum can also be obtained using a solid or liquid extract of bacteria obtained directly from a biological sample (for example, bacteria extracted from blood culture).

The embodiment shown in figure 9, allowed to test six different strains. This model can be adapted depending on the number of strains tested, in particular by increasing the number of wells.

Bibliography:

Hooff, G.P., J.J. van Kampen, R.J. Meesters, A. van Belkum, W.H. Goessens and T.M. Luider (2012). "Characterization of beta-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry." J Proteome Res 11 (1): 79-84.

Hrabak, J., V. Studentova, R. Walkova, H. Zemlickova, V. Jakubu, E. Chudackova, M. Gniadkowski, Y. Pfeifer, J.D. Perry, K. Wilkinson and T. Bergerova (2012). "Detection of NDM-1, VIM-1, cattle, OXA-48, and OXA-162 carbapenemases by matrix-assisted laser desorption ionization-time of flight mass spectrometry." J Clin Microbiol 50 (7): 2441-2443.

Hrabak, J., R. Walkova, V. Studentova, E. Chudackova and T. Bergerova (2011). "Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry." J Clin Microbiol 49 (9): 3222-3227.

Sparbier, K., S. Schubert, U. Weller, C. Boogen and M. Kostrzewa (2012). "Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against beta-lactam antibiotics." J Clin Microbiol 50 (3): 927-937.

Claims (38)

1. The method of obtaining the characterization zone of the analytical plate for at least one characterization of the population of at least one microorganism in the presence of at least one antimicrobial agent by mass spectrometry with matrix-activated laser desorption / ionization, known as MALDI, which includes the following sequentially stages: - the stage of providing an analytical plate for characterization using the MALDI method, where the plate includes at least one functionalized analytical zone carrying an antimicrobial agent; - the stage of applying a population of at least one microorganism to the specified analytical zone with contact with an antimicrobial agent; - the incubation step, which consists in maintaining the assay plate under conditions and for a time sufficient to allow the antimicrobial agent and microorganism present to interact; and - the stage of application to the analytical zone of the matrix, which is suitable for the MALDI method. 2. The production method according to claim 1, characterized in that a population of one characterized microorganism is applied. 3. The production method according to claim 1 or 2, characterized in that the applied (s) population (s) of the microorganism (s) are obtained without a preliminary step of contact with an antimicrobial agent. 4. The production method according to any one of paragraphs. 1-3, characterized in that the population (s) of the microorganism (s) are obtained after the stage of concentration, enrichment and / or purification and / or the population (s) of the microorganism (s) corresponds to (s) the colony or part of the colony obtained after growth on suitable medium, in particular gel medium. 5. The production method according to any one of paragraphs. 1-4, characterized in that the antimicrobial agent is selected so as to allow identification of the resistance of the microorganism due to the production of beta-lactamase, in particular carbapenemase. 6. The production method according to any one of paragraphs. 1-5, characterized in that the antimicrobial agent is an antibiotic, preferably selected from penicillins, cephalosporins, cefamycins, carbapenems, monobactams, in particular from ampicillin, amoxicillin, ticarcillin, piperacillin, cefuroximesime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroximesime, cefuroximetzim, , ceftriaxone, cefpodoxime, cefepime, aztreonam, ertapenem, imipenem, meropenem and faropenem. 7. The production method according to any one of paragraphs. 1-6, characterized in that it includes a functionalization step in order to obtain an assay plate for characterization using the MALDI method containing at least one assay zone carrying an antimicrobial agent, wherein said functionalization step is preferably carried out by applying an aqueous solution of an antimicrobial agent with subsequent drying to form a functionalized analytical zone. 8. The production method according to any one of paragraphs. 1-7, characterized in that the functionalized (s) analytical (s) zone (s) carries (ut) one antimicrobial agent. 9. A method for characterizing a population of at least one microorganism, where the characterization includes at least determining the presence or absence of a population of a microorganism that is resistant to at least one antimicrobial agent, characterized in that it comprises the following stages in sequence: - preparing at least one characterization zone of the analytical plate in accordance with the production method according to any one of paragraphs. 1-8; - verification of the presence of the antimicrobial agent and / or the degradation product of the antimicrobial agent using time-of-flight mass spectrometry with matrix-activated laser desorption / ionization, known as MALDI-TOF, in order to be able to conclude whether a population of a microorganism is resistant to antimicrobial agent in the characterization zone. 10. The characterization method according to claim 9, characterized in that the characterization further includes identification of the family, genus or, more preferably, the type of population of the microorganism deposited on the characterization zone. 11. The method of characterization according to claim 9 or 10, characterized in that: identify the family, gender or, preferably, the type of population a microorganism deposited on the characterization zone by conducting a first analysis by MALDI type mass spectrometry corresponding to the first stage of ionization of the characterization zone, and using the first calibration for analysis, and determine the possible presence in the characterization zone of a population of a microorganism that is resistant to the antimicrobial agent, by conducting a second analysis by mass spectrometry using the MALDI-TOF method corresponding to the second stage of ionization of the same characterization zone, and using a second calibration for analysis that differs from first calibration. 12. The method of characterization according to claim 10 or 11, characterized in that the determination of the family, genus or, more preferably, the type of population of the microorganism and determining whether the presence of an antimicrobial agent is possible, refers to the same microorganism. 13. A method for characterizing a population of at least one microorganism, wherein the characterization includes at least: identification of the family, genus, or, more preferably, the species of the microorganism population by conducting a first analysis by mass spectrometry using the MALDI method, corresponding to the first stage of ionization of the characterization zone, including the population of the microorganism (s), antimicrobial agent and matrix suitable for the MALDI method; determining the presence of a population of a microorganism that is resistant to at least one antimicrobial agent, by performing a second analysis by mass spectrometry using the MALDI method, corresponding to the second stage of ionization of the characterization zone, comprising a population of at least one microorganism, an antimicrobial agent and a matrix suitable for MALDI method characterized in that: - the first analysis and the second analysis, respectively, are carried out using a separate first stage and a separate second stage of ionization of the same characterization zone, including a population of at least one microorganism and an antimicrobial agent; - the first analysis uses the first calibration, and the second analysis uses the second calibration, which is different from the first calibration. 14. The method of characterization according to claim 13, characterized in that the applied population includes one characterized microorganism. 15. The characterization method according to claim 13 or 14, characterized in that it uses the method of obtaining the characterization zone of the analytical plate according to any one of paragraphs. 1-8. 16. An analytical plate for receiving a population of at least one microorganism to be characterized in the analytical zone using mass spectrometry using the MALDI method, characterized in that it includes an antimicrobial agent, which is applied, in particular, in the form of a solid layer to the specified analytical area of the analytical plate, forming an analytical zone, which is called functionalized. 17. The analytical plate according to claim 16, characterized in that the functionalized analytical zone is obtained by applying an aqueous solution of an antimicrobial agent, followed by drying. 18. An assay plate according to claim 16 or 17, characterized in that the antimicrobial agent is immobilized in the assay zone by electrostatic, ionic, covalent or affinity binding or, preferably, by an adhesive agent. 19. The analytical plate according to any one of paragraphs. 16-18, characterized in that the plate is formed of a polymer that contains an electrically conductive material coated with a layer of stainless steel. 20. The analytical plate according to any one of paragraphs. 16-19, characterized in that the plate contains many functionalized analytical zones, each of which carries an antimicrobial agent. 21. The analytical plate according to any one of paragraphs. 16-20, characterized in that the plate contains at least a first functionalized analytical zone carrying a first antimicrobial agent, and a second functionalized analytical zone, which is different from the first zone, carrying a second antimicrobial agent, which is different from the first antimicrobial agent. 22. The analytical plate according to any one of paragraphs. 16-21, characterized in that one or each functionalized analytical zone carries one antimicrobial agent. 23. The analytical plate according to any one of paragraphs. 16-22, characterized in that it includes one reference analytical zone, intended for subsequent intake of the population of the reference microorganism, and the fact that the surface of the reference analytical zone is free of antimicrobial agent. 24. The analytical plate according to any one of paragraphs. 16-23, characterized in that it is packed in a hermetically sealed package. 25. The use of the analytical plate according to any one of paragraphs. 16-24 in the method of characterization according to any one of the preceding paragraphs. 9-15.

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